This invention relates to a method for measuring the concentration of antigen using immunoassay, particularly to a method suitable for measuring it with high sensitivity by measuring fluorescence.
Since there was reported a method for determining a slight amount of insulin by using a specific antibody labeled with a radioisotope, this determination method called radioimmunoassay has come to be utilized for quantitating various biological substances and drugs. However, it is disadvantageous in that it uses radioisotopes, so that special care must be taken in handling them. Therefore, various kinds of immunoassays utilizing nonradioactive labels such as enzymes, substrates, fluorescent substances, chemiluminescent substances and the like are investigated, and among these, methods using an enzyme or a fluorescent substance as a label have reached the practical use stage. In particular, a fluorometric method using a fluorescent label permit determination in a short time. However, this method is also difficult to automate because its whole process requires labor and time. In order to simplify the whole process, there has been proposed a method for measuring the concentration of antigen in a sample which comprises immobilizing antibody in a membranous matrix, applying a gradient of electric potential perpendicular to the surface of the membrane, thereby moving the antigen in the measurement sample in this direction by electrophoresis, subjecting the antigen to antigen antibody reaction with the above-mentioned immobilized antibody to immobilize the same, immobilizing a label in the membranous matrix either by moving labeled antibody to the antigen immobilized by the above-mentioned procedure by electrophoresis to label the immobilized antigen, or by moving labeled antigen to the unreacted portion of the antibody immobilized in the membranous matrix by electrophoresis to label the unreacted immobilized antibody, and then measuring the concentration of the immobilized label to calculate that of the immobilized antigen. (Japanese Patent Publication Kokai (Laid-Open) No. 57257/85).
According to this method, the matrix for electrophoresis in which antigen or antibody is immobilized is in contact, on one side, with the electrolytic solution on the cathode side, and on the other side, with the electrolytic solution on the anode side, so that the time required for antigen antibody reaction which has been 3 hours to 1 day in conventional immunoassay methods can be reduced to 1 hour or less. Further, the process of conventional immunoassay can be simplified. In detail, since the unreacted substances and surplus labeled antibody penetrate the reaction membrane and move to the lower electrolytic solution, the procedure of washing with water which is necessary in immunoassay based on conventional solid-phase reaction becomes unnecessary.
However, although the above-mentioned immunoassay using electrophoresis permits simplification of the whole process and is suitable for automation of apparatus, it is disadvantageous in that when the concentration of antigen in a sample is measured by fluorometry by using antibody or antigen labeled with a fluorescent substance, scattered light and interfering fluorescence are serious.